Protein-protein interactions and nuclear trafficking of coat protein and betaC1 protein associated with Bhendi yellow vein mosaic disease

Bhendi yellow vein mosaic disease (BYVMD) is caused by a complex consisting of a monopartite begomovirus BYVMV and a satellite DNA beta component. BYVMV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction. Here we report the results of the transient expression of green fluorescent protein (GFP) fused with the betaC1 and coat protein (CP) coding regions, in the epidermal cells of Nicotiana benthamiana. GFPCP was found to be targeted into the nucleus whereas GFPbetaC1 was localized towards the periphery of the cell. The sub-cellular localization of the betaC1 protein has been compared with that of the CP in yeast cells using a genetic system for detection of protein nuclear import and export. Expression of betaC1 ORF in transgenic N. benthamiana under the control of the Cauliflower mosaic virus 35S promoter produced severe developmental abnormalities in the plant, like distorted stem, leaves and stunting of the plant. We also present the results on the interaction of CP and betaC1 proteins using yeast two hybrid analysis, suggesting a collaborative role in the inter- and intracellular dynamics of BYVMD.     

Characterization of a satellite RNA associated with cucumber mosaic virus

Two types of RNA, each with a molecular weight of approximately 0.12 × 10⁶, designated RNA 5 and satellite RNA, have been found in purified cucumber mosaic virus (CMV) preparations and have been characterized by molecular hybridization analysis using ³²P-labeled complementary DNA probes transcribed from these RNAs. RNA 5 usually makes up about 5% or less by weight of the total viral RNA and was shown to consist of specific cleavage products of CMV RNAs 1–4. Its nucleotide complexity was equivalent to about three times its molecular weight. By contrast, satellite RNA could form up to about 50% by weight of virion RNA and had the following properties: (1) It contained a unique nucleotide sequence with no homology with CMV RNAs, (2) CMV and tomato aspermy virus, but not alfalfa mosaic virus or tobacco ringspot virus, could function as helper viruses for its replication and encapsidation, (3) its nucleotide sequence was independent of the host plant and the helper virus used for its propagation and it was not derived from a host plant RNA, and (4) it was not a negative copy of any of the CMV RNA species. We concluded that this RNA is a true satellite RNA and has no relationship to the RNA found in defective interfering particles of animal viruses.     

Molecular characterization of a new alphasatellite associated with a cassava mosaic geminivirus in Madagascar

Two complete nucleotide sequences of an alphasatellite isolated from a cassava plant with mosaic disease symptoms in Madagascar are described and analyzed. While the helper begomovirus was identified as an isolate of East African cassava mosaic Kenya virus (EACMKV), its associated alphasatellite was most closely related (80 % nucleotide sequence identity) to cotton leaf curl Gezira alphasatellite. These satellite molecules have typical features of alphasatellites, with a single gene in the virion sense, an A-rich region and a stem-loop structure. According to the proposed species demarcation threshold of alphasatellites (83 % nucleotide identity), they are isolates of a new species for which we propose the name “Cassava mosaic alphasatellite”.     

Complete nucleotide sequence of two new satellite RNAs associated with cucumber mosaic virus

Complete nucleotide sequences of two new satellite RNAs (satRNA) of cucumber mosaic virus (CMV), E-satRNA and OY2-satRNA, have been determined and compared with known satellite RNA sequences. E-satRNA contained 339 nucleotide residues and OY2-satRNA had 386 nucleotide residues. Comparison of the sequences among satRNAs including these new satRNAs revealed extensive homologous regions in the 5'-terminus and the 3'-half. The former region contains about 95 nucleotide residues (80-100% homology) and the latter has about 175 nucleotide residues (81-99% homology), but there was less homology in the middle of the RNA. A possible secondary structure for E-satRNA, OY2-satRNA, and Y-satRNA [S. Hidaka et al. (1984) FEBS Lett. 174, 38-42] was constructed and compared with that of a satellite RNA from CMV-Q (Q-satRNA) [K. H. J. Gordon and R. H. Symons (1983), Nucl. Acids Res. 11, 947-960]. Two similar hairpin-loop structures in the 5'-terminal region were common features of the RNAs; one of them was found in E-satRNA, the other in OY2-satRNA, and both in Y-satRNA and Q-satRNA. Analyses of in vitro messenger properties of the satRNAs suggested that the first open reading frame of E-sat and Y-satRNAs was a possible coding region for protein synthesis.     

The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus

The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus (TM-GMV) was determined. It shows 64.4% sequence homology with the genomic RNA of tobacco mosaic virus (TMV) and 66.0% with that of tomato mosaic virus (ToMV). Its genomic organization is similar to that of TMV and ToMV. The 5' proximal open reading frame (ORF) encodes a 126K polypeptide and a 183K readthrough product in which nucleotide-binding and polymerase-sequence motifs are found. The third ORF encodes a 28.5K protein homologous to TMV and ToMV movement proteins. A conserved core is found with four other tobamoviruses and two tobraviruses suggesting a common mechanism of cell-to-cell movement for tobamo- and tobraviruses. The fourth ORF encodes the 17.5K coat protein. Homology between the RNAs of TMGMV and its satellite virus STMV is limited to their 3' termini, and structural comparisons suggest that this region may determine the nature of the satellite/helper virus interaction.     

A new satellite RNA is associated with natural infections of cucumber mosaic virus in succulent snap bean

Cucumber mosaic virus (CMV) was consistently recovered from symptomatic snap bean plants during surveys conducted in 2007 and 2008 in central Wisconsin. A large proportion of these CMV-infected plants contained a single-stranded linear RNA molecule consisting of 339 nucleotides and sharing 90–94% sequence identity with other satellite (sat) RNAs of CMV. Comparison of this satRNA sequence with currently available CMV satRNA sequences suggests this to be a novel satRNA.     

Characterization of an Indian isolate of cucumber mosaic virus infecting Egyptian henbane (Hyoscyamus muticus L.)

A cucumber mosaic virus isolate was found to be associated with mottle crinkle and severe mosaic disease of Egyptian henbane (Hyoscyamus muticus L.). The virus has been characterized as an Indian isolate of cucumber mosaic virus (CMV) based on non-persistent transmission by aphid, presence of 28-nm isometric particles, capsid protein of 26 K and single-stranded tripartite RNA genome with a subgenomic RNA (RNA 4). There was no evidence of satellite RNA genome. The isolate showed a strong serological relationship with S and A strains of CMV (CMV-S and CMV-A) in double diffusion test. A band of the 26 K capsid protein was also detected by Western blot analysis using antibodies specific to CMV-S.     

Helper virus-dependent replication, nucleotide sequence and genome organization of the satellite virus of maize white line mosaic virus

Virus like particles (17 nm in diameter) associated with maize white line mosaic virus (MWLMV) were shown to be a satellite virus of MWLMV (SV-MWLMV) on the basis of the following properties: (1) The SV-MWLMV was dependent upon the presence of MWLMV for replication in maize, while the latter virus could replicate independently of the SV particles. (2) No nucleotide sequence homology was detected between the SV-MWLMV and MWLMV, using complementary DNA probes prepared to the two RNAs, in a Northern blot hybridization analysis. (3) The RNA of the SV-MWLMV translated in vitro to produce a protein of the same Mr (24,000) as that found associated with the SV particles. This protein could be immunoprecipitated with an antiserum to the SV particles. And (4), there was no serological relationship between the coat proteins of MWLMV and the SV-MWLMV. The complete nucleotide sequence of the SV-MWLMV RNA (1168 nucleotides) was determined. The SV-MWLMV RNA contains a single open reading frame encoding a polypeptide of Mr 23,961. Computer analysis revealed no significant homology between SV-MWLMV RNA and any other viral or satellite RNAs. However, the putative SV-MWLMV capsid protein is predicted to share some structural features with the capsid protein of the satellite virus of panicum mosaic virus.     

Ubiquitinated conjugates are found in preparations of several plant viruses

Recently D.D. Dunigan, R.G. Dietzgen, J.E. Schoelz, and M. Zaitlin (Virology 165, 310-312, 1988) demonstrated that a small proportion of the subunits of tobacco mosaic virus particles were conjugated with the small protein ubiquitin. We have now detected ubiquitinated conjugates in immunoblots of virion preparations of several other plant viruses, using anti-human ubiquitin antiserum. Based on their polyacrylamide gel migrations, plant virus-associated ubiquitin-immunoreactive proteins were considered to be possible virus structural protein-ubiquitin conjugates of the following viruses: barley stripe mosaic, brome mosaic, cowpea mosaic (two proteins), cowpea severe mosaic (two proteins), and satellite panicum mosaic. Ubiquitinated conjugates were not detected in immunoblots of preparations of cucumber mosaic virus and Cymbidium mosaic virus. The significance of ubiquitinated viral proteins remains to be determined.     

Analysis of the genome of satellite panicum mosaic virus

The relatedness of the genomes of satellite panicum mosaic virus (SPMV) and its helper virus, panicum mosaic virus (PMV), were investigated by nucleic acid hybridization. The results show that the satellite and helper virus RNAs have no appreciable homology or complementarity as assessed by hybridization with cDNA probes derived from the genomes of PMV and SPMV and with a probe complementary to the 3' terminus of SPMV RNA. The complete nucleotide sequence of SPMV RNA reveals that the genome is 826 nucleotides (nt) long. The ability to label SPMV RNA with polynucleotide kinase only after phosphatase treatment suggests that the 5' terminus is phosphorylated, but the extent of phosphorylation was not determined. The first open reading frame (ORF), encountered after an 88-nt 5'-untranslated region, encodes a 17,000 mol wt protein of a size and amino acid composition that are consistent with analysis of SPMV coat protein. An additional short ORF, located near the 3' end of the RNA, could encode a 6300 mol wt polypeptide. The minus strand also contains two ORFs that could potentially encode polypeptides of 7100 and 11,000 mol wt. No evidence is available to determine whether the second positive-strand ORF or the two minus-strand ORFs are expressed. The data presented here clearly show the SPMV RNA is distinct from the RNAs of other satellite viruses, in both size and nucleotide sequence. However, the 5'-untranslated portions of SPMV and satellite tobacco mosaic virus RNAs share some structural features that may be important in initiation of translation.     

Secondary structure prediction of plant virus coat proteins

Application of the secondary structure prediction techniques to the coat protein amino acid sequences of turnip yellow mosaic virus and satellite tobacco necrosis virus yielded an excellent correlation with the patterns of secondary structural elements observed in the capsid proteins of southern bean mosaic virus and tomato bushy stunt virus. Helical predictions for the very basic N-terminal regions of three plant viruses suggested modes of protein-nucleic acid interaction similar to those proposed for histones and protamines.     

Nucleotide sequence of cucumber mosaic virus-associated RNA 5

The complete nucleotide sequence of cucumber mosaic virus-associated (satellite) RNA 5 (CARNA 5) strain D has been determined. The molecule is 335 residues long and is capped at its 5' extremity. There were minor variations in the sequence of different preparations of the satellite RNA. Otherwise, no unusual features of secondary structure or sequence are present. CARNA 5 (D) contains a number of possible initiation and termination codons for protein synthesis. Study of CARNA 5 isolated from other strains of cucumber mosaic virus suggests that sequence variation of the molecule from one strain to another is very limited.     

Bhendi yellow vein mosaic disease in India is caused by association of a DNA Beta satellite with a begomovirus

Yellow vein mosaic disease is the major limitation in the production of bhendi or okra (Abelmoschus esculentus), an important vegetable crop of India. This disease is caused by a complex consisting of the monopartite begomovirus Bhendi yellow vein mosaic virus (BYVMV, family: Geminiviridae) and a small satellite DNA beta component. BYVMV can systemically infect bhendi upon agroinoculation but produces only mild leaf curling in this host. DNA beta induces typical symptoms of bhendi yellow vein mosaic disease (BYVMD) when co-agroinoculated with the begomovirus to bhendi. The DNA beta component associated with BYVMD has a number of features in common with those reported for ageratum yellow vein disease and cotton leaf curl disease. BYVMV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction.     

A 37 kilodalton protein kinase associated with cauliflower mosaic virus

The cauliflower mosaic virus (CaMV) particle-associated protein kinase (PK) was shown to be a 37 kD protein in activity gels. In vitro experimental data concerning virus dephosphorylation or hyperphosphorylation suggested a possible regulation mechanism of this PK. The origin of the enzyme, either virus-encoded or from a host cell, is discussed.     

Complete genome sequence of a new begomovirus associated with yellow mosaic disease of Hemidesmus indicus in India

The complete DNA A genome of a virus isolate associated with yellow mosaic disease of a medicinal plant, Hemidesmus indicus, from India was cloned and sequenced. The length of DNA A was 2825 nucleotides, 35 nucleotides longer than the unit genome of monopartite begomoviruses. Comparison of the nucleotide sequence of DNA A of the virus isolate with those of other begomoviruses showed maximum sequence identity of 69 % to DNA A of ageratum yellow vein China virus (AYVCNV; AJ558120) and 68 % with tomato yellow leaf curl virus- LBa4 (TYLCV; EF185318), and it formed a distinct clade in phylogenetic analysis. The genome organization of the present virus isolate was found to be similar to that of Old World monopartite begomoviruses. The genome was considered to be monopartite, because association of DNA B and β satellite DNA components was not detected. Based on its sequence identity (<70 %) to all other begomoviruses known to date and ICTV (International Committee on Taxonomy of Viruses) species demarcating criteria (<89 % identity), it is considered a member of a novel begomovirus species, and the tentative name “Hemidesmus yellow mosaic virus” (HeYMV) is proposed.     

Molecular identification of a new begomovirus associated with yellow mosaic disease of Jatropha gossypifolia in India

Yellow mosaic disease was observed on Jatropha gossypifolia plants growing in Kathaupahadi, Madhya Pradesh, India, and whiteflies (Bemisia tabaci) were found in the vicinity. Association of a new begomovirus with yellow mosaic disease of J. gossypifolia has been detected by PCR using begomovirus DNA-A-specific primers. The complete DNA-A genome (~2.7 kb) of this virus isolate was amplified by rolling-circle amplification (RCA) followed by digestion with Bam HI. The ~2.7-kb amplicons was cloned and sequenced, and the data obtained were submitted to GenBank under accession numbers FJ177030. The genome of the virus isolate consisted of six open reading frames (ORFs): V2 (pre-coat protein) and V1 (coat protein) in the virion sense and C3 (REn protein), C2 (TrAP protein), C1 (replication-associated protein) and C4 (C4 protein) in the complementary sense. BLASTn analysis of the nucleotide sequence (2757 nt) of the viral genome (FJ177030) showed 84–85% identity and a distinct phylogenetic relationship with DNA-A of tomato leaf curl virus-Bangalore II (U38239) and tomato leaf curl Karnataka virus (AY754812). Based on its 85% sequence identity to all other begomoviruses known to date and ICTV species demarcating criteria (< 88% identity), the name Jatropha yellow mosaic India virus (JYMIV) is proposed. JYMIV is considered to be monopartite, as neither DNA-B nor DNA-β components associated with begomoviruses were detected.     

Rapid detection of the mosaic structure of the Neisseria gonorrhoeae penA Gene, which is associated with decreased susceptibilities to oral cephalosporins

In Neisseria gonorrhoeae, the mosaic structure of the penA gene (encoding penicillin-binding protein 2 [PBP 2]), which is composed of fragments of the penA genes from Neisseria cinerea and Neisseria perflava, has been significantly associated with decreased susceptibility to cephalosporins, particularly oral cephalosporins. The aim of this study was to develop a rapid assay for the detection of mosaic PBP 2 of N. gonorrhoeae by real-time PCR. This assay successfully detected the mosaic penA gene of N. gonorrhoeae, and its sensitivity was >or=10(1) copies/reaction. Six hundred twenty-one clinical strains were examined by this assay for the presence of mosaic PBP 2, which was detected in 85 (39.4%) of 216 strains from 2002, 69 (40.6%) of 170 strains from 2003, 71 (44.4%) of 160 strains from 2004, and 31 (41.3%) of 75 strains from 2005. The MICs of cephalosporins for strains with the mosaic PBP 2 detected by the assay were statistically higher than those for strains without the mosaic PBP 2. One hundred sixty-six (64.8%) of 256 strains with the mosaic PBP 2 exhibited cefixime MICs of >or=0.5 microg/ml. The emergence and spread of strains with mosaic PBP 2 could be a threat to the cefixime treatment of gonorrhea. This real-time PCR assay for the detection of mosaic PBP 2 of N. gonorrhoeae is thus useful in the prediction of decreased susceptibilities to oral cephalosporins.     

Preliminary identification and coat protein gene phylogenetic relationships of begomoviruses associated with native flora and cultivated plants from the Yucatan Peninsula of Mexico

A number of native and cultivated eudicots in the Yucatan Peninsula of Mexico (YPM) exhibit symptoms associated with virus infection. Symptomatic leaves were collected and assessed for begomoviral detection using polymerase chain reaction (PCR), and universal primers that amplify a fragment of the coat protein gene (core Cp). Begomovirus were detected in nine native and seven cultivated species, representing seven eudicot families. DNA extracts from the 16 hosts were used for PCR amplification and sequencing of a fragment containing the coat protein (Cp) gene. The complete Cp sequence was used to establish provisional species identification. Results indicated that 13 distinct begomovirus species were represented. Among these, five potentially new begomovirus species were identified, for which we propose the names Anoda golden mosaic virus (AnGMV), Boerhavia yellow spot virus (BoYSV), Papaya golden mosaic virus (PaGMV), Desmodium leaf distortion virus (DeLDV), and Hibiscus variegation virus (HiVV). Five previously described begomoviral species were provisionally identified for the first time in the YPM; these include Euphorbia mosaic virus (EuMV), Melon chlorotic leaf curl virus (MCLCuV), Okra yellow mosaic Mexico virus (OkYMMV), Sida golden mosaic virus (SiGMV), and Tobacco apical stunt virus (TbASV). Additionally, viruses previously reported from this region, Bean golden yellow mosaic virus (BGYMV), Pepper golden mosaic virus (PepGMV), and Tomato mottle virus (ToMoV) were provisionally identified in cultivated hosts. Phylogenetic analysis provisionally placed all isolates from the YPM in a Western Hemisphere begomovirus clade.     

Characterisation of a satellite RNA of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> that induces chlorosis in <Emphasis Type="Italic">Capsicum annuum</Emphasis>

The presence of Cucumber mosaic virus (CMV) satellite RNA dramatically changes symptoms on some hosts. A satellite RNA present in a strain of CMV (PepY-CMV) that induced chlorosis in pepper (Capsicum annuum) was shown to induce chlorosis in pepper in combination with another strain (Fny-CMV) that by itself induced a green mosaic symptom. The location of sequences within the PepY satellite RNA (PepY-satRNA) of CMV that conferred the ability to induce chlorosis on pepper plants were analyzed by exchanging sequence domains between cDNA clones of PepY-satRNA and an attenuated mosaic satellite RNA (Paf-satRNA), as well as site-directed mutagenesis of various clusters of the 22-nt sequence differences between the two satellite RNAs in the delimited central domain. The symptoms induced by site-directed mutants of PepY-satRNA and Paf-satRNA in the presence of Fny-CMV demonstrated an insertion within PepY-satRNA of 11 nt at positions 86–96 relative to Paf-satRNA determined the chlorosis-inducing phenotype. Within the chlorosis-inducing domain, deletion of nucleotides did not affect the satRNA replication but abolished the ability of PepY-satRNA to elicit chlorosis symptom. Conversely, a mutant satellite RNA derived from Paf-satRNA in which eleven nucleotides were inserted indicated that sequences of 11 nucleotides were found to be sufficient for chlorosis induction in pepper.